Mouse and human cDNAs encoding AES (Amino Enhancer of Split) and ESG (Enhancer of Split Groucho) proteins with strong homology to Drosophila Enhancer of split groucho protein were isolated and sequenced. Mouse AES-1 and AES-2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins were found to exhibit approximately 50% identity to the amino-terminal region of Drosophila groucho, mouse ESG and human TLE (Transducin-Like Enhancer of split) proteins. Mouse AES transcripts of 1.5Kb and 1.2Kb were abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 Kb and 1.4Kb were predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3Kb and 4.0Kb were found only in testis, while human TLE 1 transcripts of 4.5Kb were more abundant in muscle and placenta compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosome no. 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines.